EP2245047B1 - Peptides issus de pres modifiés hydrophobes du virus de l'hépatite b (vhb) et leur utilisation en tant que véhicules pour l'administration spécifique de composés au foie - Google Patents

Peptides issus de pres modifiés hydrophobes du virus de l'hépatite b (vhb) et leur utilisation en tant que véhicules pour l'administration spécifique de composés au foie Download PDF

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EP2245047B1
EP2245047B1 EP09704030.7A EP09704030A EP2245047B1 EP 2245047 B1 EP2245047 B1 EP 2245047B1 EP 09704030 A EP09704030 A EP 09704030A EP 2245047 B1 EP2245047 B1 EP 2245047B1
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liver
hbv
hydrophobic modified
derived peptide
pres
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EP2245047A1 (fr
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Walter Mier
Uwe Haberkorn
Stephan Urban
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Universitaet Heidelberg
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Universitaet Heidelberg
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/033—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6036—DNA rev transcr viruses
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to hydrophobic modified preS-derived peptides of hepatitis B virus (HBV) which are versatile vehicles for the specific delivery of compounds to the liver, preferably to hepatocytes, in vitro as well as in vivo.
  • HBV hepatitis B virus
  • This liver targeting can further be used for the targeted treatment of liver diseases or disorders, such as hepatitis, malaria, hepatocellular carcinoma (HCC), as well as for the prevention of HBV and/or HDV infection.
  • liver diseases or disorders such as hepatitis, malaria, hepatocellular carcinoma (HCC), as well as for the prevention of HBV and/or HDV infection.
  • the liver an organ which is present in vertebrates and other animals, plays a major role in the metabolism and has a number of functions in the body, including glycogen storage, decomposition of red blood cells, synthesis of plasma proteins, and detoxification.
  • the liver also is the largest gland in the human body. It lies below the diaphragm in the thoracic region of the abdomen. It produces bile, an alkaline compound which aids in digestion, via the emulsification of lipids. It also performs and regulates a wide variety of high-volume biochemical reactions requiring specialized tissues.
  • Hepatocytes make up 70 to 80% of the cytoplasmic mass of the liver. Hepatocytes are involved in protein synthesis, protein storage and transformation of carbohydrates, synthesis of cholesterol, bile salts and phospholipids, and detoxification, modification and excretion of exogenous and endogenous substances. The hepatocyte also initiates the formation and secretion of bile.
  • liver hematoma liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver hematoma, liver
  • liver disease such as biliary atresia, alpha-1-antitrypsin deficiency, alagille syndrome, and progressive familial intrahepatic cholestasis; as well as metabolic diseases.
  • Malaria which is one of the most common infectious diseases and an enormous public-health problem. Malaria is caused by protozoan parasites of the genus Plasmodium. The most serious forms of the disease are caused by Plasmodium falciparum and Plasmodium vivax, but other related species ( Plasmodium ovale, Plasmodium malariae, and sometimes Plasmodium knowlesi ) can also infect humans. This group of human-pathogenic Plasmodium species is usually referred to as malaria parasites. Malaria parasites are transmitted by female Anopheles mosquitoes. Malaria in humans develops via two phases: an exoerythrocytic (hepatic phase or "liver stage") and an erythrocytic phase.
  • the parasite is relatively protected from attack by the body's immune system because for most of its human life cycle it resides within the liver and blood cells and is relatively invisible to immune surveillance.
  • drugs that specifically address the liver specific stages of malaria parasites (e.g. primaquin) in order to obviate the development of blood stages.
  • schistosomiasis or bilharziosis, which is a parasitic disease caused by several species of flatworm. Although it has a low mortality rate, schistosomiasis can be very debilitating. It is an often chronic illness that results from infection of the blood with a parasitic flatworm (schistosome). It causes debilitation and causes liver and intestinal damage. It is most commonly found in Asia, Africa, and South America, especially in areas with water that is contaminated with fresh water snails, which contain the parasite.
  • Hepatitis B virus the cause for hepatitis B, is the prototype of a family of small, enveloped DNA viruses of mammals and birds (1).
  • the HBV envelope encloses three proteins termed L-(large), M-(middle) and S-(small) ( Figure 1A ). They share the C-terminal S-domain with four transmembrane regions.
  • the M- and L-protein carry additional N-terminal extensions of 55 and, genotype-dependent, 107 or 118 aa (preS2- and preS1) ( Figure 1B ).
  • the stoichiometric ratio of L, M and S is about 1:1:4, while the more abundantly secreted non-infectious subviral particles (SVPs) contain almost exclusively Sand only traces of L-protein (2).
  • SVPs non-infectious subviral particles
  • the preS1-domain of L is myristoylated and at some point of the HBV life cycle translocated through the ER-derived membrane. This modification is essential for infectivity (3,4).
  • HBV as well as the other hepatotropic viruses have a liver tropism, i.e. the liver is the tissue which specifically supports the growth of HBV.
  • drug targeting should fulfill the following criteria: 1) exclusive transfer of the drug to the required site of action; 2) a minimum of effects for the remaining organism; 3) use of a pharmacologically inactive vector.
  • tissue-specific enzymes for example the use of prodrugs, from which the pharmacologically active part is released in the target tissue by tissue-specific enzymes.
  • a further possibility is to couple effective, non-tissue-specific drugs to tissue-specific, but pharmacologically inert carrier systems like receptor-affine peptides or colloidal particles.
  • liver targeting of drugs has been used to enhance liver targeting of drugs.
  • a straightforward approach is based on the active phagocytosis of the reticuloendothelial system in the liver by delivering drugs in particular carriers, such as liposomes and microspheres.
  • particulate carriers incorporating a drug are mainly captured by the reticuloendothelial system in the liver, resulting in drug targeting of the liver (5).
  • liver targeting of drugs with positively charged, water-soluble polymers is based on free extravasation of most water-soluble substances from the vascular system of the liver as well as on negative charges on the liver cell surface (6).
  • polymers have been used as the carrier to allow drugs to target to the liver based on such anatomical and biochemical characteristics of the liver. More specific drug targeting of the liver has been attempted by using asialoglycoprotein receptors of liver cells.
  • the asialoglycoprotein receptor (galactose receptor) is present on hepatocytes with high density.
  • the ligand-receptor complex is internalized which allows the cellular uptake of galactosylated ligands (7).
  • HBV vectors derived from hepatitis B virus (HBV), which can be used for gene therapy, such as the delivery of therapeutic genes to liver cells and the expression of heterologous genes in liver cells.
  • HBV vectors have a limited capacity for packaging genes.
  • CN 101 045 156 A discloses "a targeting medicine" with the general formula X-L-Y, wherein X is an amino acid sequence, containing (a) an amino acid sequence derived from a protein of an infective virus with liver specificity, (b) a sequence of (a) generated by permutation, depletion, insertion or accession of one or more amino acid residues; L is an optional linker between X and Y; and Y is a medicine or radical group.
  • X can contain a polypeptide of protein pre-S1 of HBV or a variant thereof.
  • the present invention aims to provide means and methods for the effective, prevention and/or treatment of liver diseases and the regulation of the liver function.
  • this object is solved by providing a hydrophobic modified preS-derived peptide of HBV as vehicle for the specific delivery of compound(s) to the liver for use as a medicament.
  • Said hydrophobic modified preS-derived peptide has the general formula: [H m -P-R n ]A 0 .
  • P is said preS-derived peptide consisting of an amino acid sequence selected from SEQ ID NOs. 11, 12, 13, 16, 23, and 26.
  • H is said hydrophobic modification of the preS-derived peptide P, which is N-terminal of P and selected from acylation with myristoyl (C14) or stearoyl (C18); wherein m is 1.
  • R is an optional C-terminal modification (i.e. n is 0 or at least 1) of said preS-derived peptide P.
  • A is an optional anchor group (i.e. o is 0 or at least 1), which is suitable for the covalent attachment of the compound(s) and is linked to H, P and/or R.
  • Said hydrophobic modified preS-derived peptide further comprises compound(s) selected from a drug; a prodrug; a carrier or depot for a drug, or prodrug and wherein the compound(s) and the hydrophobic peptide form a conjugate.
  • this object is furthermore solved by providing the hydrophobic modified preS-derived peptide(s) of HBV of the invention for the treatment of a liver disease or disorder or for the regulation of liver function.
  • the present invention provides hydrophobic modified preS-derived peptides of hepatitis B virus (HBV) as vehicle for the specific delivery of compound(s) to the liver for use as a medicament.
  • HBV hepatitis B virus
  • HBV The envelope of HBV encloses three proteins termed L (large), M (middle) and S (small) (see Figure 1A ). They share the C-terminal S-domain with four transmembrane regions.
  • L- and M-protein carry additional N-terminal extensions of 55 and, genotype-dependent, 107 or 118 amino acids (preS2- and preS1) (see Figure 1B ).
  • preS-derived peptide of HBV refers to a peptide with an amino acid sequence that corresponds to the N-terminal extensions of the L-protein of HBV, preS1, preferably to a consensus sequence of the species and genotypes A to H as well as of woolly monkey (WMHBV), chimpanzee and gorilla hepatitis B viruses, but it also refers to variants thereof, preferably N-terminally and/or C-terminally truncated variants, amino acid substitution variants.
  • SEQ ID NO. 1 shows the HBV preS consensus amino acid sequence of the species and genotypes A to H as well as of woolly monkey (WMHBV).
  • HBV preS consensus sequence Consensus
  • A-H the eight HBV genotypes
  • WHBV woolly monkey HBV
  • HBV genotype C refers to an artificial sequence, which corresponds to or is identical to the HBV Genotype C, as e.g. shown in Genbank ABV02850.1, except that position 46 (according to the numbering as described below) is Lys (K) in the genotype C of the present invention instead of Gln (Q) as in the Genbank sequence; the HBV genotype C sequence of this application can also be referred to as "HBV genotype C Q46K". See also SEQ ID NOs. 4, 12, 21-27.
  • Figure 2 also shows the numbering of the amino acid residues of the HBV preS consensus sequence, which will be referred to throughout this specification: Amino acid residue number 1 is the methionine (Met1) of genotype D (formerly described as subtype ayw, see also SEQ ID NO. 5), whereas amino acid residue number (-11) is the methionine (Met(-11)) of genotype C (SEQ ID NO. 4).
  • Met1 or Met(-11), respectively is cleaved off by a cellular methionyl aminopeptidase and modified by a subsequent transfer of a myristoyl residue from Myristoyl-CoA to amino acid residue number 2 glycine (Gly2) or amino acid residue number (-10) glycine (Gly(-10)), respectively, by N-myristoyl transferase.
  • the N-terminal amino acid residue of genotype D is the natural amino acid Glycin (Gly2) and is numbered 2 according to the respective numbering from the codons of the underlying open reading frame of L (or e.g. Gly(-10) for genotype C).
  • the HBV preS consensus sequence also comprises the additional N-terminal amino acids of genotypes A, B, C, E, F, G and H (designated at "-" positions). Thus, in total the HBV preS consensus sequence encompasses positions (-11) to 48. Thus, there is a difference between the above described numbering and the actual listing of amino acids in SEQ ID NO. 1, e.g.
  • a hydrophobic modified preS-derived peptide of HBV provided for the use according to the present invention has the formula [H m -P-R n ]A 0 wherein
  • n and o are 0.
  • P does not comprise amino acid substitutions and/or deletions at residues 9 to 15 of SEQ ID NO. 1.
  • sequence motif NPLGFFP (corresponding to residues 9 to 15 of SEQ ID NO. 1) is not interrupted or modified, such as by replacing residues 11-15 by D-amino acids.
  • the inventors have identified these amino acid residues to be important for the liver tropism of the hydrophobic modified preS-derived peptides of HBV, as described below.
  • P consists of an amino acid sequence selected from amino acid residues 2 to 48 of the consensus sequence [SEQ ID NO. 11]; amino acid residues 2 to 21 of the consensus sequence [SEQ ID NO. 12]; amino acid residues 2 to 48 of genotype C [SEQ ID NO. 13]; amino acid residues 2 to 21 of genotype C [SEQ ID NO. 16]; amino acid residues 2 to 48 of genotype D [SEQ ID NO. 23]; and amino acid residues 2 to 21 of genotype D [SEQ ID NO. 26].
  • P does not comprise amino acid substitutions and/or deletions at residues 9 to 15 of SEQ ID NO. 1 in order to maintain the liver targeting/tropism.
  • P comprises an amino acid sequence selected from SEQ ID NO. 11 (residues 2 to 48 of the consensus sequence), SEQ ID NO. 12 (residues 2 to 21 of the consensus sequence), SEQ ID NO. 13 (residues 2 to 48 of genotype C), SEQ ID NO. 16 (residues 2 to 21 of genotype C), SEQ ID NO. 23 (residues 2 to 48 of genotype D), and SEQ ID NO. 26 (residues 2 to 21 of genotype D),
  • the hydrophobic modification (H) of the preS-derived peptide P is N-terminal of P.
  • N-terminal refers to the hydrophobic modification at the N-terminus, i.e. the respective first amino acid residue (e.g. Gly 2), but comprises also the hydrophobic modification in close proximity to the N-terminus, such as respective amino acid residues (-4), (-3), (-2), (-1), 1, 2 or 3 or 4.
  • the hydrophobic modification can furthermore be obtained by an attachment of a hydrophobic moiety at a site close to the N-terminus of P.
  • hydrophobic modification of said preS-derived peptide of HBV adds a hydrophobic moiety to the peptide.
  • m is 1, i.e. modification with one hydrophobic moiety or group.
  • hydrophobic modification of said preS-derived peptide of HBV provided for the use according to the present invention is selected from:
  • Modification by myristoylation is preferred in in vivo and medicinal applications due to its higher safety, e.g. not showing the adverse effects of the stearoyl group (innate immune response etc).
  • hydrophobic moieties is prefereably by covalent binding, which can be achieved via carbamate, amide, ether, disulfide or any other linkage that is within the skill of the person skilled in the art.
  • hydrophobic modified, namely acylated preS-derived peptides of this invention are preferably lipopeptides due to their N-terminal lipophilic or hydrophobic group/moiety.
  • the C-terminal modification (R) of said preS-derived peptide P is preferably a modification with a moiety that protects from degradation, such as in vivo degradation.
  • C-terminal refers to the modification at the C-terminus, i.e. the respective last amino acid residue, but comprises also the modification in close proximity to the C-terminus, such as the last but one amino acid residue, the last but two amino acid residue or more amino acid residues (e.g. introduction of one D-amino acid that protects the carrier from enzymatic degradation e.g. by the action of carboxypeptidases).
  • Preferred moieties that protect from degradation are selected from amides, D-amino acids, modified amino acids, cyclic amino acids, albumin, natural and synthetic polymers, such as PEG, glycane.
  • n is 0 or at least 1, i.e. the C-terminal modification (R) is optional.
  • n 1
  • n is 1, 2, 3, 4 or more. That is, the C-terminus of P or its proximity can be modified with more than one moiety or group, such as 2.
  • the moieties or groups can be the same or different to each other.
  • H and/or R are linked to P via a linker or spacer.
  • Linker or spacer are known to the skilled artisan, such as polyalanine, polyglycin, carbohydrates, (CH 2 ) n groups.
  • the anchor group (A) serves as a point of attachment for a compound, a tag, a label.
  • the anchor group is "C-terminal" of P, wherein “C-terminal” refers to the modification at the C-terminus, i.e. the respective last amino acid residue, but comprises also the close proximity of the C-terminus, such as the last but one amino acid residue, the last but two amino acid residue or more amino acid residues.
  • n can be 0, thus there is no other C-terminal modification R.
  • hydrophobic modified preS-derived peptide of HBV can have the following general formula
  • the anchor group A can be at an amino acid side chain of the preS-derived peptide P or can be the amino acid side chain of the preS-derived peptide P itself, i.e. A can be a side chain itself or a modified side chain.
  • the anchor group can also be a modified amino acid residue which was introduced into the amino acid sequence of P to serve as an anchor group.
  • anchor group A is attached to the hydrophobic modification H and/or the C-terminal modification R.
  • Preferred anchor groups are selected from ester, ether, disulfide, amide, thiol, thioester.
  • the anchor group can furthermore be suitable for attaching a complex-forming component, such as of the biotin/avidin, polyarginine/oligonucleotide (e.g. siRNA) complex.
  • a complex-forming component such as of the biotin/avidin, polyarginine/oligonucleotide (e.g. siRNA) complex.
  • o is 0 or at least 1, i.e. the anchor group (R) is optional.
  • o is 1.
  • o is 1, 2, 3, 4 or more. That is, there are more than one anchor group, such as 2.
  • the anchor groups can be the same or different to each other, allowing the attachment of several compounds, such as a drug and a label or different drugs.
  • hydrophobic modified preS-derived peptides provided for the use of the invention are the following:
  • the more preferred hydrophobic modified preS-derived peptides of the invention are: Designation of peptide Amino acid sequence Hydrophobic modification HBVpreS/2-48 stearoyl (consensus) SEQ ID NO. 11 Stearoyl (C18) HBVpreS/2-48 stearoyl (consensus) SEQ ID NO. 12 Stearoyl (C18) HBVpreS/2-48 stearoyl (C) SEQ ID NO. 13 Stearoyl (C18) HBVpreS/2-48 stearoyl (C) SEQ ID NO. 16 Stearoyl (C18) HBVpreS/2-48 myr (C) SEQ ID NO. 13 Myristoyl (C14) HBVpreS/2-48 myr (D) SEQ ID NO. 23 Myristoyl (C14) HBVpreS/2-21 stearoyl (D) SEQ ID NO. 26 Stearoyl (C18)
  • peptides of this invention can be prepared by a variety of procedures readily known to those skilled in the art, in general by synthetic chemical procedures and/or genetic engineering procedures.
  • Synthetic chemical procedures include more particularly the solid phase sequential and block synthesis (Erickson and Merrifield, 1976).
  • the solid phase sequential procedure can be performed using established automated methods such as by use of an automated peptide synthesizer.
  • an ⁇ -amino protected amino acid is bound to a resin support.
  • the resin support employed can be any suitable resin conventionally employed in the art for the solid phase preparation of (poly)peptides, preferably polystyrene which has been copolymerized with polyoxyethylen to provide sites for ester formation with the initially introduced o-amino protected amino acid.
  • This optimized method applied by the inventors, has been explicitly described (see e.g. 12).
  • the amino acids are introduced one by one (stepwise).
  • Each synthesis cycle corresponding to the introduction of one amino acid includes a deprotection step, successive washing steps, a coupling step with activation of the amino acid, and subsequent washing steps. Each of these steps is followed by a filtration.
  • the reactive agents for coupling are the classical reactive agents for (poly)peptide synthesis such as dicyclohexylcarbodiimide, hydroxybenzotriazole, benzotriazil-1-yl-oxytris (dimethylamino) phosphonium hexafluorophosphate, and diphenylphosphorylazide.
  • the polypeptide is separated from the resin by a treatment with a strong acid such as trifluoroacetic acid in the presence of anisol, ethanedithiol or 2-methylindole.
  • a strong acid such as trifluoroacetic acid in the presence of anisol, ethanedithiol or 2-methylindole.
  • the compound is then purified by the classical techniques of purification, in particular by means of HPLC.
  • the peptides of the present invention may also be obtained by coupling (poly)peptide fragments that are selectively protected, this coupling being effected e.g. in a solution.
  • the peptides can further be produced by genetic engineering techniques as known to the skilled artisan.
  • An eukaryotic expression system such as the baculovirus system, is particularly suitable. According to this procedure proteins are expressed in insect cells infected with a recombinant baculovirus containing a nucleic acid sequence encoding a heterologous protein and regulating nucleic acid sequences, such as a promoter.
  • a recombinant baculovirus containing a nucleic acid sequence encoding a heterologous protein and regulating nucleic acid sequences, such as a promoter.
  • Several cell-lines are available for infection with recombinant baculovirus, such as cell line Sf-9, available from the American Type Culture Collection (CRL 1711).
  • Expression in prokaryotic expression system such as E. coli, is also particularly suitable.
  • hydrophobic moiety to the peptide can be accomplished by a variety of procedures readily known to those skilled in the art, including synthetic and genetic engineering approaches.
  • the peptides and/or fusion peptides can be produced by stably transfected eukaryotic cell lines, like CHO and other cell lines which are known in the art and usually used for generating vaccines and the like. Due to the intrinsic property that the N-terminal 47-preS1 amino acids promote secretion of a myristoylated protein/peptide, the biologically active hydrophobic modified peptide can be extracted from cell culture supernatants.
  • the present invention provides the hydrophobic modified preS-derived peptides of HBV as vehicle or shuttle for the specific delivery of a compound to the liver for use as a medicament.
  • “Vehicle” or “shuttle” for the specific delivery of a compound to the liver according to the present invention refers to the liver tropism or hepatotropism of the hydrophobic modified preS-derived peptides of HBV as found by the inventors and described herein, i.e. to their capacity to selectively accumulate in the liver, preferably to selectively accumulate at the plasma membrane of hepatocytes as well as to selectively enter into hepatocytes.
  • the invention is based on the finding of a highly specific liver accumulation and on the identification of the determinants of the liver tropism of HBV in the preS 1 sequence of HBV by the inventors.
  • the invention uses the knowledge about the determinants of the liver tropism for the design of universal vehicles or shuttles for specific liver targeting or delivery, respectively.
  • hydrophobic modified preS-derived peptides of the present invention are versatile vehicles or shuttles for specifically delivering compound(s) to the liver.
  • the specific delivery of a compound to the liver is the specific delivery of the compound to hepatocytes.
  • the compound can specifically be delivered to hepatocytes in vitro as well as in vivo.
  • the compound is preferably specifically delivered to the liver of an animal, preferably mammal or human, or a bird.
  • the "compound” to be specifically delivered to the liver according to this invention are selected from drugs; prodrugs; a carrier or depot for a drug, or prodrug.
  • Drugs can be in form of prodrugs or preprodrugs.
  • the compound to be specifically delivered to hepatocytes is a drug (or a drug in form of a prodrug) and is preferably selected from Drug class Examples interferon IFN ⁇ viral reverse transcriptase inhibitor Lamivudine, Adefovir, Entecavir viral RNA polymerase inhibitor HCV NS5B polymerase inhibitor viral core assembly inhibitor or for hepatropic viruses viral nucleocapsid inhibitor e.g.
  • dihydroarylpyrimidine-derived HBV capsid assembly inhibitors kinase inhibitor Raf kinase inhibitor Sorafenib (BAY 43-9006) nucleoside analogue protease inhibitor HCV NS3 protease inhibitor, BILN-2061, VX950 - viral or general protease inhibitor proteasom inhibitor MG132, bortezomib antibody or fragment thereof Neutralizing anti-HCV E2 antibodies siRNA or precursor thereof HBV, HCV, HDV-specific siRNAs and modifications thereof; siRNAs targeting other sequences, including cellular sequences farnesylation inhibitor; such as farnesyl transferase inhibitor (FTI) alcohol dehydrogenase (ADH) or activator thereof cholesterol biosynthesis inhibitor, such as HMD-CoA-reductase inhibitors Mevinacor/Lovastatin inhibitor of the liver stage of a virus or a non-viral pathogen, such as malaria, schistosomiasis (bilharzio
  • the compound is preferably selected from a clinically approved drug.
  • the compound to be delivered is IFN-alpha.
  • P preferably comprises an amino acid sequence of SEQ ID NO. 23 and/or H is a hydrophobic modification with myristoyl (C14).
  • the hydrophobic modified preS1-derived peptide(s) used in such an embodiment can be HBVpreS/2- 20 myr (D), HBVpreS/2-48 myr (D), HBVpreS/2-20 stearoyl (D), HBVpreS/2-48 stearoyl (D) (see also Figure 14 ).
  • a fusion protein or construct with mouse interferon-alpha was obtained by recombinant expression in insect cells using the baculovirus expression system. See also Figures 14 and 15 as well as Examples.
  • the compound is interferon, such as IFN ⁇
  • P preferably comprises an amino acid sequence of SEQ ID NO. 23 and/or H is a hydrophobic modification with myristoyl (C14)
  • the compound is labelled, i.e. carries a label as defined herein.
  • the compounds are in form of depots or carriers, which e.g. carry a drug, prodrug.
  • depots or carriers are known in the art, such as nanoparticles, liposomes, microbubbles, gas emulsions.
  • the compound and the hydrophobic modified preS-derived peptide of HBV form a conjugate.
  • the conjugate of compound and hydrophobic modified preS-derived peptide is formed by covalent attachment or by complex formation.
  • the compound is covalently attached to the hydrophobic modified preS-derived peptide, preferably by attaching the compound to an anchor group A.
  • the form of attachment depends on the type of compound. The person of skill in the art will be able to determine suitable anchor groups for forming suitable conjugates.
  • A furthermore comprises a spacer or linker.
  • the spacer or linker preferably comprises a recognition site for hepatocyte specific activation, which is preferably recognized by a liver protein.
  • the recognition site is preferably a proteolytic cleavage site.
  • the liver protein is, thus, preferably a hepatocellular protein, more preferably a hepatocellular proteolytic enzyme.
  • the conjugate can be administered to a subject and will be transported through the body, such as in the bodily fluids, without being cleaved. However, as soon as the conjugate reaches its target, the liver or the hepatocytes, respectively, the liver protein, such as a hepatocellular proteolytic enzyme will cleave the proteolytic cleavage site and release the compound from its shuttle, i.e. the hydrophobic modified preS-derived peptide.
  • liver proteins are cytochromes, such as cytochrome P450.
  • the HepDirect® technology (of Metabasis Technologies, Inc.) as used in Adefovir or Pradevofir, is also suitable for the present invention.
  • the conjugate of compound and hydrophobic modified preS-derived peptide is formed by complex formation.
  • Preferred complexes useful in the invention are biotin/avidin, polyarginine/oligonucleotide (e.g. siRNA). The skilled artisan will be able to determine suitable complex components and to design the compound and hydrophobic modified preS-derived peptide accordingly.
  • the hydrophobic modified preS-derived peptide in particular the conjugates of the present invention, are preferably used to enrich a compound, that is shuttled to the liver, in the liver.
  • the compound is cleaved of the conjugate with the hydrophobic modified preS-derived peptide of HBV by a liver protein, preferably a hepatocellular proteolytic enzyme, in particular in vivo in the liver.
  • the above hydrophobic modified preS-derived peptides in particular their conjugates with compounds, are provided for the treatment of a liver disease or disorder.
  • the respective compound is selected and selectively, specifically delivered to the liver.
  • liver disease or a “liver disorder” according to the present invention refers to any disease or disorder that has an effect on or involves the organ liver, liver tissue or hepatocytes.
  • liver diseases are:
  • liver diseases of animals such as pets or livestock
  • diseases that can be transmitted to humans such as toxoplasmosis
  • the liver disease or disorder to be treated is preferably selected from hepatitis, cirrhosis, haemochromatosis, preferably hepatitis caused by hepatitis A, B, C, D, E, F, G and H virus.
  • the liver disease or disorder to be treated can also be concomitant hepatitis caused by viruses, such as viruses of the family Herpesviridae, e.g. herpes virus, cytomegalie virus (CMV) but also varicella zoster virus (VZV), Epstein Barr virus (EBV), coxsackie viruses, yellow fever virus, Dengue virus.
  • viruses of the family Herpesviridae e.g. herpes virus, cytomegalie virus (CMV) but also varicella zoster virus (VZV), Epstein Barr virus (EBV), coxsackie viruses, yellow fever virus, Dengue virus.
  • the liver disease or disorder to be treated can also be a disease which involves a liver stadium of a virus or a non-viral pathogen, such as in many tropical diseases. Since the liver stadium of some pathogens is an early stadium, the respective infection can be selectively, specifically treated in such an early stadium.
  • viruses are hepatitis A, B, C, D, E, F, G and H virus, herpes viruses.
  • Such non-viral pathogens are bacteria, parasites and/or worms.
  • Parasites are for example protozoan parasites of the genus Plasmodium that cause malaria, such as Plasmodium falciparum, Plasmodium vivax, and related species (e.g. Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi ).
  • Such worms are for example flatworms of the genus Schistosoma that cause schistosomiasis or bilharziosis, such as Schistosoma mansoni, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum and Schistosoma mekongi.
  • Such parasites are also for example Leishmania trypanosome protozoa of the genus Phlebotomus and Lutzomyia which are responsible for the disease leishmaniasis.
  • malaria malaria, schistosomiasis (bilharziosis), and/or leishmaniasis can be treated by the means of this invention.
  • liver disease or disorder to be treated are preferably liver tumors, preferably hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • the liver disease or disorder to be treated can also be a metabolic disease, such as diabetes, hyperlipidemia, metabolic syndrome and obesity, chronic hyperglycemia, metabolic syndrome, non-alcoholic steatohepatitis (NASH) (see also (9)).
  • a metabolic disease such as diabetes, hyperlipidemia, metabolic syndrome and obesity, chronic hyperglycemia, metabolic syndrome, non-alcoholic steatohepatitis (NASH) (see also (9)).
  • NASH non-alcoholic steatohepatitis
  • the above hydrophobic modified preS-derived peptides in particular their conjugates with compounds, are provided for the regulation of liver function.
  • the compound to be delivered to the liver is preferably an immunogenic epitope.
  • the above described hydrophobic modified preS-derived peptides are also suitable for the prevention of hepatitis B and/or D infection.
  • This is possible due to the property of the above described hydrophobic modified preS-derived peptides to target not only the liver (liver tropism) but furthermore act as viral entry inhibitors of HBV and/or HDV, which can be seen e.g. in Figure 9 .
  • the above described hydrophobic modified preS-derived peptides are suitable HBV and/or HDV vaccines.
  • acylated preS-derived peptides in particular their conjugates with compounds, can be provided for the combined treatment of a liver disease or disorder and the prevention of hepatitis B and/or D infection.
  • the use according of the invention comprises the use of a pharmaceutical composition comprising at least one hydrophobic modified preS-derived peptide of HBV as defined herein and at least a compound to be specifically delivered to the liver as defined herein and optionally a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition according to the present invention comprises:
  • compositions according to the present invention are very well suited for all the uses and methods described herein.
  • a “pharmaceutically acceptable carrier or excipient” refers to any vehicle wherein or with which the pharmaceutical or vaccine compositions according to the invention may be formulated. It includes a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, and standard pharmaceutical practice.
  • the present disclosure describes methods for the diagnosis, prevention and/or treatment of a liver disease or disorder by utilizing the hydrophobic modified preS-derived peptide(s) of HBV or the pharmaceutical composition(s) of the invention.
  • the present disclosure also describes a method for the combined diagnosis, prevention and/or treatment of a liver disease or disorder and the prevention of HBV and/or HDV infection by administering to a subject a conjugate as defined herein, which comprises a hydrophobic modified preS-derived peptide of HBV and a compound, or a pharmaceutical composition as defined herein.
  • the method for the combined diagnosis, prevention and/or treatment of a liver disease or disorder and the prevention of hepatitis B and/or D infection can comprise administering to a subject in a therapeutically effective amount
  • the route of administration of the conjugates or pharmaceutical compositions of the present invention is selected from subcutaneous, intravenous, oral, nasal, intramuscular, transdermal, inhalative, by suppository.
  • a preferred embodiment for nasal administration or application is a nasal spray.
  • a “therapeutically effective amount” of a conjugate or a pharmaceutical composition of this invention refers to the amount that is sufficient to treat the respective liver disease or disorder and/or to prevent hepatitis B and/or D infection.
  • a preferred therapeutically effective amount is in the range of 10 ⁇ g to 1 mg per kg body weight.
  • the preferred therapeutically effective amount is in the range of 10 ⁇ g to 1 mg per kg body weight.
  • a preferred therapeutically effective amount is about 100 ⁇ g per kg body weight or in the range of 1 to 5 mg per patient.
  • the preferred therapeutically effective amount depends on the respective compound that is to be delivered and its respective therapeutically effective amount.
  • the disclosure also describes the preS-derived peptides, i.e. nucleic acids encoding them, in gene therapy approaches.
  • a viral vector comprises a nucleic acid encoding a preS-derived peptide P as defined herein for the gene therapy of a liver disease or disorder.
  • the nucleic acid encodes the amino acid sequence of the preS-derived peptide P, which is necessary and sufficient to accomplish liver targeting.
  • the viral vector is replication defective, preferably an adeno-associated viral vector.
  • the viral vector can further comprise a heterologous sequence to be expressed in the liver.
  • the viral vector can be a gene therapeutically suitable vector, which are known to the skilled person.
  • the disclosure further describes a method for in vitro and/or in vivo identification of a hepatocyte receptor involved in the attachment and/or penetration of HBV and/or quantitation of the expression of said receptor that comprises using a hydrophobic modified preS-derived peptide as described above.
  • Said hepatocyte receptor can be identified in mammals or respective animal models, such as mouse or human.
  • said method comprises the steps comprising:
  • the method for the identification of a HBV receptor comprises the use of
  • Enzyme labels comprise conjugation of an enzyme to a molecule of interest, e. g. a polypeptide, and can be detected by any of colorimetric, spectrophotometric, or fluorospectrophotometric techniques.
  • HBV Hepatitis B Virus
  • Hallmarks of HBV infection are a remarkable in vivo efficacy and a pronounced liver tropism. The latter is probably the result of a specific interaction of one of the three HBV surface proteins with liver associated receptors or liver-targeting factors in the circulation system.
  • the present invention led to the development of a new class of peptide-drug conjugates to target liver diseases, such as HCC, and optionally to interfere with HBV and HDV infection.
  • HBV-preS1-surface protein-derived lipopeptides that efficiently block HBV entry in vitro and in vivo.
  • Biodistribution studies of the present invention on these inhibitory peptides revealed that they selectively accumulate in the liver were they bind to and presumably enter into hepatocytes.
  • This hepatotropism requires N-terminal acylation of the peptide and depends on a certain HBV preS-sequence motif within the N-terminal 47 preS1 amino acids, i.e. within the amino acid residues 2 to 21 or amino acid residues 2 to 20 (or preferably the minimal sequence of residues 9 to 15).
  • this peptide sequence additionally bears a membrane translocation signal which facilitates the transport of even complete fusion proteins across plasma membranes (unpublished results) opens the possibility of specifically delivering any kind of drug to the plasma membrane of hepatocytes or selectively even into this cell.
  • HBV preS1-derived lipopeptides are capable to completely prevent HBV infection in a transplanted uPA-RAG-1 mouse model at very low doses.
  • Pharmakokinetic studies on these HBVpreS-derived entry inhibitors indicated a remarkable hepatotropism combined with an extraordinary high serum stability (t 1/2 ca. 60 h) and a long half life time in the target organ (t 1/2 ca. 24 h).
  • Both N-terminal acylation as well as the integrity of the certain amino acid sequence of the peptides are mandatory (i.e. within the amino acid residues 2 to 21 (or preferably the minimal sequence of residues 9 to 15)).
  • the peptides can be used as versatile vectors for liver specific drug targeting to conquer infections of hepatocytes or to treat hepatocellular carcinoma.
  • the inventors have furthermore proven the principle that WMHBV infection can be efficiently blocked through subcutaneous application of HBV envelope protein-derived peptides in vivo. This opens new perspectives for the prevention of acute HBV-infection and therapeutic options for chronic hepatitis B. Since the uPA/RAG-2/Pfp mice used lack B cells, T cells, and NK cells, a direct inhibitory effect of the peptides on susceptible hepatocytes is assumed. This is supported by the efficient accumulation of acylated preS-derived peptides in the liver, followed by a slow clearance possibly via the biliary route. Both properties permit subcutaneous application at very low doses and low frequencies.
  • the biodistribution of the hydrophobic modified preS-derived peptides was studied in male NMRI mice. All experiments were performed in compliance with German laws.
  • the peptides, containing an additional Tyr-residue at the C-terminal end were labelled with 131 I (Amersham Biosciences, Freiburg, Germany) by the chloramine-T method and purified by HPLC.
  • the labelled peptides were subcutaneously administered by injection of a solution in 50% DMSO.
  • mice were sacrificed and the radioactivity contained in the blood, heart, lung, spleen, liver, kidney, muscle and brain was measured in a ⁇ -counter (Canberra Packard, Rüsselsheim, Germany) and expressed as a percentage of injected dose per gram of tissue (%ID/g).
  • I labelled HBVpreS/2-48 myr D was extracted from one liver lobe 24 h post subcutaneous injection. To that aim, 1 ml water per gram frozen liver tissue was added to the sample. After homogenization an equal volume of acetonitrile was added and the homogenization was repeated. After centrifugation (2 x 10 min at 4000 x g) this solution was separated on a reverse phase HPLC column and the radioactivity of each fraction was quantified in a gamma counter.
  • HepaRG cells were grown in William's E medium supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 100 ⁇ g/ml streptomycin, 5 ⁇ g/ml insulin and 5x10 -5 M hydrocortisone hemisuccinate (10). Cells were passaged 1/5 every two weeks by trypsination. Two to three weeks before infection cell differentiation was induced by adding 2% DMSO into the maintenance medium. The medium was exchanged every 2-3 days.
  • FCS fetal calf serum
  • HepG2 clone 2.2.15 (11) cells As an infectious inoculum, a 50-fold concentrated culture supernatant of HepG2 clone 2.2.15 (11) cells was used, because of an unlimited supply and a constant quality. It was prepared from freshly collected supernatants by precipitating viral particles in the presence of 6% polyethylene glycol (PEG) 8000. The pellet was resuspended in phosphate buffered saline (PBS) containing 25% FCS. Aliquots were stored at -80°C. Differentiated HepaRG cells or PHH were incubated with the concentrated infectious source, 10-fold diluted in culture medium supplemented with 4% PEG 8000 (Sigma), for 20 h at 37°C. At the end of the incubation, cells were washed three times with the culture medium and maintained in the presence of 2% DMSO and 5x10 -5 M hydrocortisone hemisuccinate and harvested at indicated times.
  • PEG polyethylene glycol
  • HepaRG cells were infected either in absence (0 nM) or in the presence of 1, 5, 25, 100 and 2000 nM of the respective peptides of the invention.
  • the infectious inoculum (HBV of genotype D) and the peptides were incubated overnight. After washing, cells were maintained for another 12 days to allow viral gene expression.
  • Cell culture supernatants from day 8-12 were collected and analyzed for secreted HBSAg using a quantitative commercially available ELISA.
  • HBsAg values from the respective uncompleted infection were set to 100 % and the degree of infection inhibition is given in % of the uncompleted infection.
  • Cryo-preserved primary hepatocytes were thawed and washed with serum-free medium.
  • 4 x 10 5 cells/ml were incubated with the respective peptide at a concentration of 200 nM in serum-free cell culture medium. The staining was performed for 30 minutes at room temperature with frequent mixing. Subsequently, cells were washed extensively and resuspended in PBS to proceed with the FACS analysis.
  • HBVpreS1-peptide - mouse interferon alpha fusion proteins were expressed in Hi5-insect cells using the baculovirus expression system.
  • the fusion proteins secreted in the cell supernatants were harvested on day 5 post infection and purified in a first step by an affinity chromatography for the His-tag fused C-terminally to the preS1-interferon.
  • the activity of the preS1-interferon proteins in the elution fractions was measured by their ability to inhibit Newcastle disease virus mediated cell death.
  • the elution fractions containing functional preS1-interferon proteins were further purified by gel filtration chromatography on a S75-sepharose column with a 1M urea-PBS buffer (see Figure 15 ).

Claims (14)

  1. Peptide issu de préS modifié hydrophobe du virus de l'hépatite B (VHB) possédant la formule

            [Hm-P-Rn]A0

    en tant que véhicule pour l'administration spécifique de composé(s) au foie, destiné à être utilisé en tant que médicament,
    dans laquelle
    P est ledit peptide issu de préS consistant en une séquence d'acides aminés sélectionnée parmi SEQ ID NO: 11, 12, 13, 16, 23, et 26 ;
    H est ladite modification hydrophobe du peptide P issu de préS, qui est l'extrémité N-terminale de P et sélectionnée parmi une acylation avec un stéaroyle (C18) ou un myristoyle (C14),
    m est 1,
    R est une modification C-terminale dudit peptide P issu de préS,
    qui est de préférence un fragment qui confère une protection contre la dégradation, sélectionné parmi un amide, un acide D-aminé, un acide aminé modifié, un acide aminé cyclique, l'albumine, un polymère naturel et synthétique, tel que le PEG, un glycane,
    n est 0 ou au moins 1,
    A est un groupe d'ancrage,
    de préférence sélectionné parmi un ester, un éther, un disulfure, un amide, un thiol, un thioester,
    o est 0 ou au moins 1,
    comprenant en outre un/des composé(s) sélectionné(s) parmi un médicament ; un promédicament ; un véhicule ou un dépôt pour un médicament, ou un promédicament, et où le(s) composé(s) et le peptide hydrophobe forment un conjugué.
  2. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon la revendication 1, dans lequel H et/ou R sont liés à P via un segment de liaison ou un espaceur.
  3. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon la revendication 1, dans lequel le composé est sélectionné parmi un médicament, de préférence
    un interféron, tel que l'IFNα ;
    un inhibiteur de transcriptase inverse virale ;
    un inhibiteur d'ARN polymérase virale ;
    un inhibiteur d'assemblage du noyau viral ou un inhibiteur de nucléocapside virale ;
    un inhibiteur de kinase, tel qu'un inhibiteur de kinase Raf ;
    un analogue de nucléoside ;
    un inhibiteur de protéase ;
    un inhibiteur du protéasome ;
    un anticorps ou un fragment de celui-ci ;
    un ARNsi ou un précurseur de celui-ci ;
    un inhibiteur de farnésylation ; tel qu'un inhibiteur de la farnésyltransférase (FTI) ; l'alcool déshydrogénase (ADH) ou un activateur de celle-ci ;
    un inhibiteur de la biosynthèse du cholestérol ;
    un inhibiteur du stade hépatique d'un virus ou d'un agent pathogène non viral ;
    un antibiotique,
    où, de préférence, le médicament est sous la forme d'un promédicament.
  4. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon la revendication 3, dans lequel le composé est un interféron, tel que l'IFNα, et P comprend de préférence une séquence d'acides aminés de SEQ ID NO: 23 et/ou H est une modification hydrophobe avec un myristoyle (C14).
  5. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon l'une quelconque des revendications 1 à 4, dans lequel le conjugué est formé par un attachement covalent ou par la formation d'un complexe, de manière davantage préférée par un attachement covalent d'un composé à un groupe d'ancrage A.
  6. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon l'une quelconque des revendications 1 à 5, dans lequel le groupe d'ancrage A comprend en outre un espaceur ou un segment de liaison,
    dans lequel l'espaceur ou le segment de liaison comprend de préférence un site de reconnaissance, qui est de préférence reconnu par une protéine hépatique, de préférence une protéine hépatocellulaire, de manière davantage préférée une enzyme protéolytique hépatocellulaire ou un cytochrome.
  7. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon l'une quelconque des revendications 1 à 6, comprenant l'utilisation d'une composition pharmaceutique comprenant :
    au moins un peptide issu de préS modifié hydrophobe du VHB selon la revendication 1 ou 2 ;
    au moins un composé devant être spécifiquement administré au foie, de préférence à des hépatocytes, de préférence tel que défini dans l'une quelconque des revendications 1, 3 à 4 ;
    ou un conjugué tel que défini dans la revendication 5 ; et
    facultativement un véhicule et/ou excipient pharmaceutiquement acceptable.
  8. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon l'une quelconque des revendications 1 à 7, dans lequel l'administration spécifique d'un composé au foie est de préférence l'administration spécifique du composé à des hépatocytes.
  9. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon la revendication 8, dans lequel le composé est spécifiquement administré au foie d'un animal, de préférence un mammifère ou un humain, ou un oiseau,
    et/ou dans lequel le composé est de préférence enrichi dans le foie,
    et/ou dans lequel le composé est de préférence clivé du conjugué covalent avec le peptide issu de préS modifié hydrophobe du VHB par une protéine hépatique, de préférence une protéine hépatocellulaire, de manière davantage préférée une enzyme protéolytique hépatocellulaire ou un cytochrome.
  10. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon l'une quelconque des revendications 1 à 9, destiné à être utilisé dans un procédé de traitement d'une maladie ou d'un trouble hépatique.
  11. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon la revendication 10, dans lequel la maladie ou le trouble hépatique est sélectionné(e) parmi l'hépatite, la cirrhose, l'hémochromatose, de préférence l'hépatite provoquée par le virus de l'hépatite A, B, C, D, E, F, G et H ou une hépatite concomitante provoquée par des virus,
    et/ou dans lequel la maladie ou le trouble hépatique est une maladie qui implique un stade hépatique d'un virus ou d'un agent pathogène non viral, de préférence une maladie tropicale, la malaria, la schistosomiase, la leishmaniose.
  12. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon la revendication 10, dans lequel la maladie ou le trouble hépatique est une tumeur hépatique, de préférence un carcinome hépatocellulaire (CHC),
    ou dans lequel la maladie ou le trouble hépatique est une maladie métabolique, de préférence l'obésité, le syndrome métabolique, la stéatohépatite non alcoolique (NASH).
  13. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon la revendication 10, destiné à être utilisé dans la régulation de la fonction hépatique et/ou destiné à être utilisé dans la présentation d'un antigène médiée par un hépatocyte et l'activation de réponses immunologiques dirigées vers le foie.
  14. Peptide issu de préS modifié hydrophobe destiné à être utilisé selon la revendication 10 dans la prévention d'une infection par l'hépatite B et/ou D en tant que vaccin.
EP09704030.7A 2008-01-25 2009-01-26 Peptides issus de pres modifiés hydrophobes du virus de l'hépatite b (vhb) et leur utilisation en tant que véhicules pour l'administration spécifique de composés au foie Active EP2245047B1 (fr)

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WO2008098182A1 (fr) 2007-02-08 2008-08-14 University Of Utah Research Foundation Procédés et compositions apparentés à l'inhibition d'une entrée virale
US8875361B2 (en) 2008-05-21 2014-11-04 Wirtz Manufacturing Co., Inc. Reformed battery grids
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CN102241744B (zh) * 2010-05-14 2015-03-04 上海贺普药业股份有限公司 一种病毒感染阻断剂、其药物组合物及其应用
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JP6486003B2 (ja) * 2011-02-10 2019-03-20 ルプレヒト−カールス−ウニヴェルジテート ハイデルベルクＲｕｐｒｅｃｈｔ−Ｋａｒｌｓ−Ｕｎｉｖｅｒｓｉｔａｅｔ Ｈｅｉｄｅｌｂｅｒｇ 肝臓特異的診断のための疎水性修飾ペプチド
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JP2011512123A (ja) 2011-04-21
WO2009092612A1 (fr) 2009-07-30
CN102015753A (zh) 2011-04-13
EP2245047A1 (fr) 2010-11-03
US9868768B2 (en) 2018-01-16
JP5686468B2 (ja) 2015-03-18
US20110027183A1 (en) 2011-02-03

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